HRP PROCEDURE -TMB

1. Survival = 20-24 h
2. Perfuse with buffered saline, then fixative, then sucrose- postfixative
3. Remove brain, block, place in 10% sucrose 6 h to 4 days
4. Cut 20-50 urn frozen sections, collect in phosphate buffer
5. Mount sections onto subbed slides and air dry
6. Reaction media 40 min
7. Reaction media + substrate = 50 min
8. D'H20 wash x6 -30 sec each
9. 70% EtOH = 30-45 sec
10. 95% EtOH = 60 sec
11. 100% EtOH = 2X 30 sec
12. Xylene = 2X 2 min (max. 20 min total)
13. Coverslip
14. Examine unstained sections w/ bright & dark field
15. Select sections to stain
16. Float off coverslips in xylene
17. Re-hydrate through steps 11, 10 and 9 to D'H2O
18. Neutral Red stain = 2.5 min
19. D'H2O = 5-10 sec rinse
20. Dehydrate through steps 9,10 and 11
21. Xylene = 2X 2 min
22. Coverslip